Polymorphism and DNA markers for asparagus cultivars identified by random amplified polymorphic DNA

Summary DNA polymorphism among five Asparagus officinalis L. cultivars-Imperial, Snow, Steline, UC-157 and Larac, as detected by random amplified polymorphic DNA (RAPD), is reported. Thirty one decamer primers were tested. and twenty six of them yielded amplification products. Fourteen primers gave products with at least one polymorphic DNA fragment. Among a total of 119 amplified fragments 33 were polymorphic. These RAPD markers enabled the identification of asparagus cultivars. Unique markers for cultivars were: Snow-bands 475 bp, 772 bp, 412 bp, 935 bp and 820 bp amplified by primers D5, OPA-07, OPA-09, OPA-10 and OPA-18, respectively. Steline-bands 645 bp, 680 bp and 997 bp amplified by primers A32, OPA-03 and OPA-09, respectively. A band 903 bp, amplitied by primer OPA-12, is a marker for Imperial, and a band 420 bp, amplified by primer D52, is a marker for Larac. Cultivar UC-157 could be identified by a combination of shared polymorphic bands. The pairwise marker difference between cultivars ranged from 0.08 to 0.17. A phenogram of the genetic relationship based on RAPD fits with the known origin of the cultivars.     

Variation in Capsicum annuum revealed by RAPD and AFLP markers

Genetic relationships were examined among thirty-four pepper (Capsicum annuum) cultivars of different types. Two types of PCR-based markers were used, RAPD and AFLP, and their relative effectiveness was compared. A dendrogram based on RAPD markers separated the large-fruited sweet cultivars from the small-fruited pungent peppers, and the former group showed less divergence than the latter. The percentage of polymorphic markers was lower for AFLP than for RAPD markers (13 and 22% respectively). However, AFLP primers amplified on average six times more products than RAPD markers. The average numbers of polymorphic products per primer were 1.6 and 6.5 for RAPD and AFLP primers, respectively, i.e., AFLP primers were four times more efficient than RAPD primers in their ability to detect polymorphism in pepper. While four blocky type cultivars were indistinguishable by RAPD, two AFLP primer pairs were sufficient to distinguish the four cultivars from each other. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vine-1 retrotransposon-based sequence-specific amplified polymorphism for Vitis vinifera L. genotyping

The sequence‐specific amplification polymorphism (S‐SAP) method, derived from the amplified fragment length polymorphism (AFLP) technique, produces amplified fragments containing retrotransposon long terminal repeat ( LTR ) sequence at one end and a host restriction site at the other. The development and application of this procedure to the LTR of the Vine‐1 element from grapevine is reported. Two primers derived from one of the LTR sequences flanking the retrotransposon were used in combination with MseI degenerated primers on 15 grapevine accessions. S‐SAP results were compared with AFLP data. The heterozygosity and gene diversity values were higher for S‐SAP than for the AFLP procedure. Results show that S‐SAP amplification is effective in identifying polymorphisms and defining genetic distances among cultivars, and could be used for fingerprinting and for ‘Traminer’ clone identification. To the contrary Vine‐1 retrotransposon‐based S‐SAP was not able to distinguish ‘Pinot’ clones.     

Random amplified polymorphic DNA variation among German and Dutch asparagus cultivars

DNA polymorphism among nine cultivars of Asparagus officinalis L. was measured using random amplified polymorphic DNA (RAPD). Of 69 reproducible amplification products from 12 arbitrary decamer primers, 49 RAPD markers were polymorphic and could be used to distinguish six German and three Dutch asparagus cultivars. Even with very small sample sizes, genetic similarity measurements based on the RAPD data allowed accurate grouping of the nine cultivars into distinct clusters, with the exception of two individuals which clustered to closely related varieties. Two German cultivars showed high genetic similarity and were distinct from the remaining German varieties. The German and Dutch cultivars were clearly separated by a relatively large genetic distance.     

Genetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs

In order to obtain an overview of the genetic diversity present within the set of pea cultivars released in Germany, 21 cultivars were analysed at the DNA level by random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs), as well as for agronomic traits. Yield of grain cultivars ranged from 2.95 to 3.87 t/ha. Based on the screening of 60 RAPD primers and 32 Eco RI + 3/Mse I+3 AFLP primer combinations, 20 RAPD primers and 11 Eco RI + 3/MseI+ 3 primer combinations generating polymorphic and distinct fragments were chosen for estimation of genetic diversity. Twenty RAPD primers amplified a total of 314 scorable bands ranging from about 262 bp to 1996 bp. Of these, 175 fragments (55.7%) were polymorphic. Based on these data, genetic similarity (GS) was estimated between 0.80 (‘Lisa’ vs.‘Grapis’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’; mean GS = 0.88). Eleven AFLP primer combinations led to the amplification of 949 scorable fragments ranging from 43 to 805 bp and of these, 462 (48.7%) were polymorphic. Genetic similarity based on AFLPs was calculated between 0.85 (‘Lisa’ vs.‘Laser’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’, mean GS = 0.90). Correlation of genetic similarity estimated on RAPDs and AFLPs was estimated at r = 0.79** using Spearman's rank correlation coefficient and at r = 0.84 by the Mantel test, respectively. UPGMA cluster analysis carried out on these data separately for RAPDs and AFLPs and on the combined data reflected, to some extent, pedigree relationships and cophenetic correlations (r = 0.89 for RAPDs, r = 0.88 for AFLPs, and r = 0.93 RAPDs + AFLPs) indicate a good fit of respective clusters to genetic similarity data. The correlation of cluster analyses to pedigree information and the impact on parental genotype selection is discussed.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

Comparing RAPD and AFLPTM analysis in discrimination and estimation of genetic similarities among apple (Malus domestica Borkh.) cultivars

Forty-one apple (Malus × domestica Borkh.) cultivars were screened for RAPD (Random Amplified Polymorphic DNA) and AFLP(Amplified Fragment Length Polymorphism) markers. RAPD analysis was performed with 35 arbitrary 10-mer primers, selected from 60 primers tested (kits A, C and E, Operon Technologies, Inc.). Of a total of 362bands observed, 208 (57.5%) were polymorphic. Three-hundred-and-eighty-one AFLP fragments were obtained with 8primer combinations, of which 218 (57.2%) were polymorphic. Cultivars differentiated through mutation were included in this study and showed identical patterns when analysed with both RAPD and AFLP analysis. The estimated genetic relationships were correlated (r = 73.7%) between the analysis with the two different markers. UPGMA analysis was performed and dendrograms were constructed using either the data apart from each(RAPD and AFLP) method or combined in a single joint matrix. The relationships among the forty-one studied cultivars were basically consistent with the known lineage and geographic origins of the cultivars. The four Portuguese cultivars included in this study clustered together and diverged from the other cultivars. Apparently they constitute an independent genetic pool, which could be of interest for apple plant breeders. This revised version was published online in July 2006 with corrections to the Cover Date.     

Clustering of amplified fragment length polymorphism markers in a linkage map of rye

Amplified fragment length polymorphisms (AFLPs) are now widely used in DNA fingerprinting and genetic diversity studies, the construction of dense genetic maps and in fine mapping of agronomically important traits. The AFLP markers have been chosen as a source to extend and saturate a linkage map of rye, which has previously been generated by means of restriction fragment length polymorphism, random amplified polymorphic DNA, simple sequence repeat and isozyme markers. Gaps between linkage groups, which were known to be part of chromosome 2R, have been closed, thus allowing the determination of their correct order. Eighteen EcoRI‐MseI primer combinations were screened for polymorphism and yielded 148 polymorphic bands out of a total of 1180. The level of polymorphism among the different primer combinations varied from 5.7% to 33.3%. Eight primer combinations, which revealed most polymorphisms, were further analysed in all individuals of the F₂ mapping population. Seventy‐one out of 80 polymorphic loci could be integrated into the linkage map, thereby increasing the total number of markers to 182. However, 46% of the mapped AFLP markers constituted four major clusters located on chromosomes 2R, 5R and 7R, predominantly in proximity to the centromere. The integration of AFLP markers caused an increase of 215 cM, which resulted in a total map length of almost 1100 cM.     

Genotyping safflower (Carthamus tinctorius) cultivars by DNA fingerprints

Summary Carthamus tinctorius (2n = 2x = 24) (family Asteraceae), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia and the Americas as a source of high-quality vegetable and industrial oil. India ranks first in the production of safflower oil. Fourteen cultivars, widely cultivated in various agro-climatic regions of India, have been fingerprinted by RAPD, ISSR, and AFLP markers utilizing 36, 21 primers, and 4 primer combinations, respectively. On an individual assay basis, AFLP has proven to be the best marker system as compared with the other two markers applied as assessed by high discriminating power (0.98), assay efficiency index (33.2), marker index (18.2), resolving power (40.62), and genotype index (0.856). Thirty-six RAPD and 21 SSR primers could differentiate a maximum of eight and four cultivars, respectively, whereas, two AFLP primer combinations could fingerprint all the 14 cultivars. To understand genetic relationships among these cultivars, Jaccard's similarity coefficient and UPGMA clustering algorithm were applied to the three marker data sets. Mean genetic similarities ranged from 0.689 (AFLP) to 0.952 (ISSR). Correlation coefficient comparisons between similarity matrices and co-phenetic matrices obtained with the three markers revealed that AFLP displayed no congruence vis-a-vis RAPD and ISSR data. However, strong correlation was observed between RAPD and ISSR marker systems. This paper reports the start of molecular biology programme targeting nuclear genome of safflower, a major world oilseed crop about whose genetics very little is known.     

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.     

Evaluation of amplified fragment length polymorphism markers in tef, Eragrostis tef (Zucc.) Trotter, and related species

Tef is an important cereal crop in Ethiopia. This study was conducted to investigate (1) genetic diversity within and among three Eragrostis species (E. tef, E. pilosa and E. curvula), and (2) the relationship between E. tef, E. pilosa and E. curvula. A total of 529 AFLP markers were obtained, out of which 58% (368) were polymorphic, using 10 primer, combinations. The three species were separated distinctly using amplified fragment length polymorphism (AFLP), However, diversity revealed at the morphological trait level was not commensurate with that observed for AFLP. This was due to the small number of morphological traits available and their interaction with the environment. Within tef, ‘Rubicunda’ and DZ‐01‐1093 were found to be distantly related to the rest of the tef accessions. The diversity within species was such that E. pilosa was the most diverse followed by E. curvula and E. tef. Moreover, E. pilosa was more closely related to E. tef than E. curvula. Therefore, further study is needed of E. pilosa accessions and of ‘Rubicunda’ and DZ‐01‐1093 in a crossing programme to generate a population for selection and/or genetic mapping. A total of 19 cultivars or accessions had one or more unique fragments using one or more AFLP primers, indicating the potential of the technology in fingerprinting tef in a breeding or seed multiplication programme.     

Cultivar identification and genetic relationship among selected breeding lines and cultivars in chickpea (Cicer arietinum L.)

Twenty two RAPD and 22 ISSR markers were evaluated for their potential use in determination of genetic relationships in chickpea (Cicer arietinum L.) cultivars and breeding lines. We were able to identify six chickpea cultivars/breeding lines by cultivar-specific markers. All of the cultivars tested displayed a different phenotype generated either by the RAPD or ISSR primers. Though ISSR primers generated less markers than RAPD primers, the ISSR primers produced higher levels of polymorphism (% of polymorphic markers per primer) than RAPD primers. A high level of within cultivar homogeneity was observed in chickpea. Cultivars/breeding lines originating from a common genetic background showed closer genetic relationship. Chickpea lines with similar seed type(kabuli or desi) had a tendency to cluster together. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purity control of F1-hybrid tomato cultivars by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were applied in purity control of hybrid seed production of tomato (Lycopersicon esculentum Mill.). DNA from three commercial F₁-hybrid cultivars and their parental lines was subjected to RAPD screening with 50 primers. Two of four primers which detected polymorphism between the parents tested, generated paternal-specific RAPDs, enabling a clear distinction to be made between hybrids and their maternal parents. In addition, combination of the polymorphic DNA products generated by these primers exhibited hybrid-specific patterns, enabling each cultivar to be identified. This result indicates the practical usefulness of RAPD markers in hybrid-tomato-seed purity-control tests and cultivar identification. The approach is advantageous in its rapidity and simplicity, particularly as an alternative for those cultivars for which lengthy and costly phenotypic tests are currently used.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

Identification of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP

The identification of an amplified fragment length polymorphism (AFLP) marker linked to an agronomically useful trait in sesame is reported. A bulked segregant analysis (BSA) approach was adopted on segregating progenies of a cross between the closed capsule mutant line ‘cc3’, and the Turkish variety ‘Muganli‐57′. A total of 72 primer combinations were screened for linkage to the trait, but only one closely linked amplified fragment length polymorphism (AFLP) marker was identified. The linkage was confirmed by analysing the AFLP profile from single plants. The marker has the potential to accelerate breeding programmes aimed at modifying unwanted side‐effects of the closed capsule mutation by marker‐assisted selection.     

Analyses of genetic diversity in Cuban rice varieties using isozyme, RAPD and AFLP markers

A survey of the genetic diversity among the major cuban rice cultivars was conducted using isozyme, RAPD and AFLP markers. Polymorphisms were detected for esterases, peroxidases, alcohol dehydrogenases and polyphenoloxidases systems; 21 RAPD primers and four AFLP primer combinations. Heterozygosity arithmetic mean value (H_av(p)), the effective multiplex ratio (EMR) and the marker index (MI), were calculated for isozyme, RAPD and AFLP markers. The mean value of genetic similarity among the different varieties was 0.92 for isozyme, 0.73 for RAPD and 0.58 for AFLP analyses. Thus, AFLP were able to detect polymorphisms with higher efficiency than RAPD (+15%) and isozyme (+34%). Data from the isozyme, RAPD and AFLP analyses were used to compute matrices of genetic similarities. The efficiency of the UPGMA for the estimation of genetic relatedness among varieties was supported by cophenetic correlation coefficients. The resulting values indicated that the distortion level for the estimated similarities was minimal. The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for isozyme and RAPD markers (r = 0.89), as well as for AFLP and RAPD markers (r = 0.82) were properly related. However, AFLP and isozyme data showed only moderate correlation (r = 0.63). Although the genetic variability found among the different cultivars was low, both RAPD and AFLP markers proved to be efficient tools in assessing the genetic diversity of rice genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP-based STS markers closely linked to a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley

The Rfm1 gene restores the fertility of the msm1 and msm² male‐sterile cytoplasms in barley. Rfm1 is located on the short arm of chromosome 6H. To develop molecular markers tightly linked to Rfm1 for use in sophisticated marker‐assisted selection and map‐based cloning, an amplified fragment‐length polymorphism (AFLP) marker system with isogenic lines and a segregating BC₁F₁ population was used. Nine hundred primer combinations were screened and a linkage map was constructed around the Rfm1 locus by using 25 recombinant plants selected from 214 BC₁F₁ plants. Three AFLP markers were identified, e34m2, e46m19 and e48m17, linked to the locus. The most closely linked markers were e34m2, at 1.0 cM distally and e46m19, at 1.1 cM proximally. The two AFLP markers were converted to dominant STS markers. These markers should accelerate programmes for breeding restorer lines and will be useful for map‐based cloning.     

Relationship between heterosis and genetic distance based on morphological traits and AFLP markers in pepper

Genetic diversity is considered as one of the criteria for the selection of parents for hybrid breeding. The present study was undertaken to evaluate genetic divergence among seven pepper cultivars and to assess the relationship between heterosis and parental genetic distance. Twenty‐one F₁ hybrids and seven parents were evaluated for 15 morphological characters in a greenhouse and in the field. The parents were examined for DNA polymorphisms using six amplified fragment length polymorphism (AFLP) primer combinations. Cluster analysis using two genetic distance measures grouped the seven parents differently. Mid‐parent and high‐parent heterosis was observed for most characters. Most hybrids outperformed the parental lines for fruit yield, earliness and plant height. Morphological and AFLP‐based distance measurements were efficient enough to allocate pepper genotypes into heterotic groups. The correlations of morphological distances with mid‐parent heterosis were significant for days to flowering and maturity, suggesting earliness can be predicted from morphological distances of parental lines. However, the correlations of AFLP‐measured genetic distances with mid‐ and high‐parent heterosis were non‐significant for all characters, except for fruit diameter, and proved to be of no predictive value.     

The use of AFLP markers for the identification of carrot breeding lines and F1 hybrids

Four inbred lines of carrot (cytoplasmic male‐steriles and corresponding maintainers) and eight of their F₁ hybrids were studied with the amplified fragment length polymorphism (AFLP) technique to examine their genetic relationship and produce markers useful for testing hybrid seed purity. Eighty‐six polymorphic amplicons were identified in bulked DNA samples using eight primer pair combinations. Genetic distance was estimated on the basis of the presence or absence of polymorphic bands. The dendrogram plotted on the basis of the AFLP data closely represented the pedigree relationships of the lines and their hybrids. From one to six amplicons specific for a breeding line were identified. Most of them were also present in the DNA bulks of respective F₁ hybrids. However, screening performed on individual plants of two parental lines and the corresponding hybrid indicated insufficient uniformity of parental lines, limiting the applicability of AFLP markers for testing hybrid seed purity.